Interleukin-16 (IL-16) was first described in 1982 as lymphocyte chemoattractant factor (LCF; Center, et al., (1982) J. Immunol. 128: 2563–2568; Cruikshank, et al., (1982) J. Immunol. 128: 2569–2574). Subsequent studies showed that IL-16 is a multifunctional cytokine, selectively inducing the migration of CD4+ T cells, eosinophils and monocytes. IL-16 also acts as a growth factor for resting CD4+ T cells, promoting their entry into the G1 phase of the cell cycle, and inducing interleukin-2 receptor and major histocompatibility (MHC) class II protein expression on the cell surface. These activating functions are associated with intracellular signals including the synthesis of inositol trisphosphate (IP3) and a transient increase of intracellular Ca2+ concentration (reviewed in Cruikshank, et al. (1998) International Reviews of Immunology 16: 523–540).
CD4 is a ˜55 kDa type 1 integral cell surface glycoprotein with four extracellular immunoglobulin-like extracellular domains (D1–D4), a single membrane-spanning region, and short intracytoplasmic tail which interacts with intracellular tyrosine kinases such as p56lck. The extracellular domains appear to form two rigid structures consisting of D1D2 and D3D4, with a flexible connection between D2 and D3 (Brady, et al., (1993) Science 260: 979–983). The D1D2 domain interacts with MHC class II proteins and a high affinity (Kd ˜10 nM) HIV-1 gp120 binding site maps to the N-terminus of the V1 region of CD4, overlapping with but distinct from the MHC class II binding site (Fleury, et al., (1991) Cell 66: 1037–1049). The seminal plasma gp17 binding site on CD4 is also located in the D1 domain, close to, but distinct from the gp120 binding site (Autiero, et al., (1997) European Journal of Biochemistry 245: 208–213).
CD4 is a receptor for IL-16. IL-16 induces chemotactic responses in CD4+ but not CD4− T lymphocytes Berman, et al. (1985) 95:105–112. The T cell chemoattractant response to IL-16 is inhibited by co-incubation with Fab fragments of the anti-CD4 monoclonal antibody OKT 4, and the magnitude of the IL-16-induced cell migration by monocytes is directly proportional to the amount of CD4 expressed on the responding cells (Cruikshank, et al., (1987) J. Immunol. 138: 3817–3823). In addition, transfection of human CD4 confers IL-16-responsiveness to an otherwise unresponsive L3T4− murine hybridoma cell line as demonstrated by the induction of cell motility and rises in intracellular Ca2+ and IP3 which are inhibited by OKT4 Fab (Cruikshank, et al., (1991) J. Immunol. 146: 2928–2934).
Surface expression of CD4 is required for cells to respond to IL-16, and a direct interaction between IL-16 and CD4 was observed in co-immunoprecipitation experiments (Cruikshank, et al. (1998) International Reviews of Immunology 16: 523–540)). The CD4 ligand HIV-1 envelope glycoprotein gp120 and certain anti-CD4 antibodies mimic some of the bioactivities of IL-16 (Kornfeld, et al., (1988) Nature 335: 445–448; Ledbetter, et al., (1987) Proc. Natl. Acad. Sci. USA 84: 1384–1388; and Neudorf, et al., (1990) Cell. Immunol. 125: 301–314). Certain chemokine receptors are known to function as co-receptors with CD4 for HIV-1 infection (Feng, et al., (1996) Science 272: 872–877; Dragic, et al., (1996) Nature 381: 667–673), but it is unknown whether co-receptors are utilized by IL-16. Another soluble CD4 ligand was reported by Autiero et al. (Autiero, et al., (1991) Experimental Cell Research 197: 268–271; Autiero, et al., (1995) Eur. J. Immunol. 25:1461–1464) who isolated a human seminal plasma glycoprotein, gp17, which binds to recombinant soluble CD4 coupled to Sepharose beads as well as to CD4+ Jurkat cells. The physiological role of gp17 is presently unknown. Together, these findings indicate that CD4 is multi-functional receptor.
There is also direct physical evidence for an IL-16-CD4 interaction. IL-16 can be co-immunoprecipitated with recombinant soluble CD4, and rIL-16 partially displaces OKT4 bound to CD4 (Cruikshank, et al. (1994) Proc. Natl. Acad. Sci. USA 91: 5109–5113). Data from several laboratories indicate a high degree of sequence and functional homology for IL-16 across different animal species (Bannert, et al., (1998) Immunogenetics 47: 390–397; Keane, et al., (1998) J. Immunol. 160: 5945–5954; Leutenegger, et al., (1999) Molecular cloning and expression of feline interleukin-16, (UnPub)).
Human IL-16 induces chemotaxis of human, rat, and mouse CD4+ T cells (Center, D. M. and Cruikshank, W. W. (1982) J. Immunol. 128: 2563–2568). Murine IL-16 also induces motility and interleukin-2 receptor (IL-2R)-expression in human and murine target cells. It is therefore believed that the site(s) on CD4 interacting with IL-16 are also likely to be conserved. Comparison of the predicted amino acid sequences of CD4 across several species indicates that the D4 domain of CD4 is critical for IL-16 bioactivity.
CD4 is also the major receptor for human immunodeficiency virus-1 (HIV-1), HIV-2, and human herpes virus-7 (Dalgleish, et al., (1984) Nature 312: 763–767; Klatzmann, et al. (1984) Nature 312: 767–768; Lusso, et al., (1994) Proceedings of the National Academy of Sciences of the United States of America 91, 3872–3876). Originally identified as a differentiation antigen on T lymphocytes, CD4 was later found to be expressed on a variety cell types including monocytes, macrophages, eosinophils, hematopoietic progenitor cells, neurons, and spermatoza (Foti, et al., (1995) Journal of Laboratory & Clinical Medicine 126: 233–239). Expression of CD4 by these non-lymphocytic cells indicates that it mediates functions independent of the T cell antigen receptor, although the nature of these putative functions remain to be defined. In addition to binding MHC class II proteins, it is believed that CD4 can serve as a receptor for other soluble ligands.
Until recently, no function has been attributed to the D4 domain of CD4. Wu et al. (Wu, et al., (1997) Nature 387: 527–530) reported the x-ray crystallographic structure of recombinant soluble human D3D4 which spontaneously dimerizes at high concentration in solution. Wu et al. found that domain 4 (D4) mediates CD4 dimerization, and that the interface between dimers involves D4 domains exclusively. At the center of the interface is a pair of conserved glutamine residues (Gln345 and Gln345′) separated by a hydrogen-bonding distance. In their model, the level of CD4 expression when evenly distributed on a cell surface (estimated at ˜10−5 M) would favor monomers. During antigen recognition, CD4 recruited by cooperative interactions at the cell-cell adhesion junction would lead to an increased local concentration favoring dimer formation. Wu et al. proposed that CD4 dimerization-mediated trans autophosphorylation is required for CD4-associated kinase activation, and subsequent intracellular signaling. In support of this model, Satoh et al. (Satoh, et al., (1996) Biochemical & Biophysical Research Communications 224: 438–443) found that D4-based peptides were capable of inhibiting a mixed lymphocyte reaction (MLR). The activity of these peptides was postulated to result from competitive binding to CD4, thus inhibiting CD4 dimerization.
Comparison of the human CD4 amino acid sequence with that of several different species revealed that immunoglobulin-like domain 4 (D4) is the most conserved extracellular region. A comparison of the amino acid sequence of the human CD4 D4 domain with the CD4 D4 domain of mice reveals that 37 out of 73 amino acids are identical. Mouse and human D4 regions have an amino acid sequence homology of approximately 63% as determined by the method of Lipman et al., (1985) Science 227: 1435–1441.
As it is established that IL-16 is a key modulator of immune and inflammatory diseases, it would be desirable to identify IL-16 antagonists, i.e., substances capable of blocking or interrupting the activity of IL-16, for use in anti-inflammatory compositions in the treatment of, e.g., asthma, rheumatoid arthritis or inflammatory bowel disease. Such compositions may also prove to be more advantageous over presently available NSAIDS, steroid based anti-inflammatory drugs and cytotoxic drugs which often have severe side effects with the continued usage that is required for chronic inflammatory diseases.